ARTICLE
Auteur(s) : Didem
Didar Balci1, Nizami Duran2, Burcin
Ozer2, Ramazan Gunesacar3, Yusuf
Onlen4, Julide Zehra Yenin1
1Mustafa Kemal University, Faculty of Medicine,
Department of Dermatology, 31100 Hatay, Turkey
2Mustafa Kemal University, Faculty of Medicine,
Department of Microbiology and Clinical Microbiology,
Hatay, Turkey
3Mustafa Kemal University, Faculty of Medicine,
Department of Medical Biology and Genetics, Hatay,
Turkey
4Mustafa Kemal University, Faculty of Medicine,
Department of Infectious Disease, Hatay, Turkey
Article reçu le 21 Janvier 2009, accepté le 21 Janvier 2009
Psoriasis is a T cell-dependent autoimmune skin disease
characterized by remission and exacerbation. The disease affects
approximately 2% of the general population in Europe [1]. Activated
T lymphocytes play an important role in the development and
maintenance of psoriatic plaques. Stimulation of dendritic cells
and macrophages, which are called antigen-presenting cells, results
in the activation of Th cells. These differentiate into IFN-γ,
producing Th1 cells, and IL-17, producing Th17 cells. Interaction
of these cells with macrophages, mast cells and neutrophils results
in cytokine releasing and inflammation, leading to keratinocyte
proliferation [2]. Recently, a T cell subset population has been
identified, T regulatory cells, whose role is to suppress
inflammatory responses triggered by T effector cells. A recent
study demonstrated significantly lower proliferation and secretion
levels of the cytokines IL-2 and IL-10 from regulatory T cells in
response to streptococcal superantigen (Strep-A) in psoriasis,
compared with healthy controls [3].
Superantigens are bacterial and viral proteins which bind to
class II major histocompatibility complex (MHC) molecules and Vβ
segments of the T cell receptor, resulting in T cell activation and
cytokine release. Streptococcal and staphylococcal infections have
been suspected as triggering and exacerbating factors in psoriasis.
Some strains of Staphylococcus aureus (S. aureus) and Streptococcus
sp. produce toxins that are called superantigens [4]. The
application of staphylococcal superantigen toxic shock syndrome
toxin (tst-1) and staphylococcal enterotoxin (se) b onto the skin
of psoriasis patients demonstrated a greater inflammatory response
than that in the skin of normal subjects or atopic dermatitis or
lichen planus patients [5]. Psoriasis and atopic dermatitis
represent the most frequent chronic inflammatory skin diseases, and
have some similarities and some differences. The aggravating role
of S. aureus superantigens is well known in atopic dermatitis [4,
6]. Yamamoto et al. demonstrated an increased hyper-reactivity of
peripheral blood to superantigens in patients with psoriasis and
suggested that superantigens may lead to the exacerbation and
persistence of psoriasis [7]. In the literature, sea, seb, sec,
sed, exfoliative toxin (et) and tst-1 have been shown to trigger
exacerbation of psoriasis. However, the association between the
presence of S. aureus superantigens and the disease severity of
psoriasis remains controversial [8-10].
In this case-control study, we have evaluated the association of
se a through e, eta and etb, tst and methicillin resistance gene
(mecA) with psoriasis. We have also evaluated if the S. aureus
colonization and the presence of superantigens are correlated with
the disease severity.
Material and methods
Study subjects
Fifty consecutive patients with chronic plaque-type psoriasis (age,
42.2 ± 15.7 year) attending our dermatology outpatient clinic and
50 sex- and age-matched healthy controls (age, 43.3 ± 15.3 year)
were included in this study. Informed consent was obtained from all
participants and the study protocol was approved by the ethics
committee of our institution. The diagnosis of plaque psoriasis was
based on a clinical or histopathological examination of all
patients. Exclusion criteria for all study subjects were:
immunodeficiency, nasal rhinitis or other inflammatory disease,
nasal steroid use or history of sino-nasal surgery. Subjects who
had other inflammatory dermatological disorders or systemic
diseases or had been treated with systemic or topical antibiotics
in the previous 4 weeks or had been hospitalized in the previous 2
months were also excluded from the study.
The duration of the disease and medication of the patients were
recorded. At the time of the study, 21 patients were taking topical
corticosteroids, 1 was taking topical calcipotriol, 8 were taking a
combination of topical corticosteroid and calcipotriol, 7 were
taking systemic acitretin and 1 was taking etanercept. The
remaining 12 patients were not receiving any treatment. None of the
patients was taking systemic immunosuppressive drugs. The disease
severity was evaluated using the Psoriasis Area and Severity Index
(PASI).
Isolation of S. aureus
Skin swab samples were taken from psoriatic plaques for lesional
skin of psoriasis patients for the isolation of staphylococci. Skin
swab samples were taken from the volar site of the elbow for
non-lesional skin and normal skin in psoriasis patients and healthy
control subjects, respectively. Also, nares swab samples were
obtained from both nares in psoriasis patients and the control
group. To take the swab samples, the skin and lesional areas were
briefly wiped to remove any extraneous organic matter and
subsequently disinfected with a suitable disinfectant (70% alcohol)
to remove contaminating bacteria and the other residues such as
ointments. A sterile cotton swab was dipped in
double-distilled water and then a gentle swabbing of the skin and
the anterior nares of the subjects was passed repeatedly over the
affected skin area.
The samples were applied on 10% blood agar plates (Biomerieux,
France) and incubated at 37 °C for 24 hours. S. aureus was
identified by testing typical colonies for coagulase activity [11].
Staphylococcus strains were stored at – 70 °C in
Mueller-Hinton broth (Merck, Germany) supplemented with 40%
glycerol (v/v).
DNA Isolation
For nucleic acid isolation, samples of frozen Staphylococcus were
thawed, and all strains were subcultured in brain-heart infusion
broth (Merck, Germany) overnight with shaking at 37 °C. Total
DNA was isolated from 5 mL of a broth culture grown overnight
for all S. aureus strains used in the study. The procedure used for
DNA isolation has been described previously by Johnson et al. [12].
According to this method, bacterial cells were harvested from the
cultures by centrifugation at 3.000 × g for 10 min. And then
the cell pellet was re-suspended in phosphate-buffered saline with
100 μg of lysostaphin (Sigma) per mL, and incubated at
37 °C for 30 min. Phenol-chloroform extractions were used
for nucleic acid extraction from all samples, and DNA was
precipitated with ethanol. The precipitate was dissolved in 50 μL
of TE buffer (10 mM Tris chloride-1 mM EDTA [pH 8.0], and
stored at – 20 °C until processing. The primers used for
S. aureus toxin genes were those reported [12-14] and are listed in
table 1.
The polymerase chain reaction (PCR) amplification was performed
in a 25 μL reaction mixture. The PCR was performed under the
following parameters: The reaction mixture consisted of 2.5 mL
of 10× reaction buffer without MgCl2 (Promega Corp.); 200 μM of
each deoxynucleoside triphospate [AB Gene, UK], 3 mM MgCl2;
20 pmol of primers for sea, seb, sec and see; 40 pmol of
primers for sed, and approximately 10 ng of template DNA, and
brought up to a 25 μL final volume with distilled water.
Reactions were hot started for 5 min at 94 °C and
placed on ice, and 1 U of Taq polymerase (Fermentas, USA) was
added. Each sample was subjected to 35 PCR cycles, consisting of
94 °C for 2 min, 2 min at 57 °C and 1 min
at 72 °C. A final elongation step at 72 °C for
7 min was also applied in a thermal cycler (Bioder/Thermal
Blocks xp cycler, Tokyo Japan).
Furthermore, for tst, eta, etb and mecA genes, the following
amplification cycles were performed for the combination of primer
sets for tst, eta, etb and mecA. Denaturation for 2 min at
94 °C, annealing of primers for 2 min at 55 °C, and
primer extension for 1 min at 72 °C with autoextension.
The cycles were terminated by a final extension step at 72 °C for
5 min. (The reaction mixture consisted of 2.5 mL of 10×
reaction buffer without MgCl2, 200 μM each of deoxynucleoside
triphospate [AB Gene, UK], 3 mM MgCl2; 20 pmol of primers for
tst, etb and mecA; 40 pmol of primers for eta). The PCR products
were analyzed with gel electrophoresis by 2% agarose and visualized
using a gel imaging system (Wealtec, Dolphin-View, USA) (figures 1 and 2).
Table 1 Nucleotide sequences, locations within the
genes, and predicted sizes of PCR products for the S. aureus
toxin-specific oligonucleotide primers used in this study
|
Gene
|
Primer
|
Oligonucleotide sequence (5’-3’)
|
Location of gene
|
Size of amplified product (bp)
|
|
sea
|
sea-1
|
TTG GAA ACG GTT AAA ACG AA
|
490-509
|
120
|
|
sea-2
|
GAA CCT TCC CAT CAA AAA CA
|
591-610
|
|
seb
|
seb-1
|
TCG CAT CAA ACT GAC AAA CG
|
634-653
|
478
|
|
seb-2
|
GCA GGT ACT CTA TAA GTG CC
|
1091-1110
|
|
sec
|
sec-1
|
GAC ATA AAA GCT AGG AAT TT
|
676-695
|
257
|
|
sec-2
|
AAA TCG GAT TAA CAT TAT CC
|
913-932
|
|
sed
|
sed-1
|
CTAGTTTGGTAATATCTCCT
|
354-373
|
317
|
|
sed-2
|
TAATGCTATATCTTATAGGG
|
652-671
|
|
see
|
see-1
|
AGG TTT TTT CAC AGG TAC TCC
|
237-257
|
200
|
|
see-2
|
CTT TTT TTT CTT CGG TAC ATC
|
425-445
|
|
tst
|
tst-1
|
ATG GAC GAC TCA GCT TGA TA
|
251-270
|
350
|
|
tst-2
|
TTT CCA ATA ACC ACC CGT TT
|
581-600
|
|
eta
|
eta-1
|
CTA GTG CAT TTG TTA TTC AA
|
374-393
|
119
|
|
eta-2
|
TGC ATT GAC ACC ATA GTA CT
|
473-492
|
|
etb
|
etb-1
|
ACG GCT ATA TAC ATT CAA TT
|
51-70
|
200
|
|
etb-2
|
TCC ATC GAT AAT ATA CCT AA
|
231-250
|
|
mecA
|
mecA-1
|
ACTGCTATCCACCCTCAAAC
|
1182–1201
|
163
|
|
mecA-2
|
CTGGTGAAGTTGTAATCTGG
|
1325–1344
|
Statistical analysis
Results were expressed as mean ± standard deviation (SD) for
continuous data and percentages for categorical data. Test of
normality of the variables were performed using the
Kolmogorov-Smirnov test. Patients and control subjects were
compared using Student’s t-test for continuous variables and the
chi-square test for categorical variables. In order to show the
relations between the PASI scores and both S. aureus cultivation
and toxin production in the lesional skin, non-lesional skin and
nares of the patients with psoriasis, Spearman rank correlation
analysis was used. The relationship was analyzed between PASI
scores and two-sided P values of less than 0.05 were considered
statistically significant. The statistical analysis was carried out
using the Statistical Package for the Social Sciences (SPSS)
version 11.0 (SPSS, Chicago, IL, USA).
Results
S. aureus was cultivated from the nares in 25 (50%) of 50 patients
with psoriasis and in 17 (34%) of 50 healthy controls (p >
0.05). The numbers of S. aureus strains isolated from the
non-lesional skin of the patients and from the normal skin of the
healthy controls were 7 (14%) and 6 (12%), respectively (p >
0.05). S. aureus was cultivated from lesional skin in 32 (64%) of
50 patients with psoriasis. There was a statistical difference in
the cultivation of S. aureus between lesional (64%) and
non-lesional skin (14%) in patients with psoriasis (p = 0.037).
Thirty one (96.8%) out of the 32 strains isolated from the
lesional skin and 3 (42.3%) out of the 7 strains isolated from the
non-lesional skin were toxigenic (p = 0.01). None of the strains
isolated from the normal skin in healthy controls was toxigenic
whereas 3 (42.3%) out of the 7 strains isolated from the
nonlesional skin in patients with psoriasis were toxigenic (p >
0.05). Strains isolated from the nares were toxigenic in 96%
(24/25) for patients with psoriasis and in 41.2% (7/17) for healthy
controls, respectively (p = 0.006). The distribution of
superantigens in the study subjects is shown table 2.
Patients who were cultivation-positive in lesional skin had a
significantly higher PASI score than patients who were
cultivation-negative in lesional skin (8.28 ± 3.97 vs. 5.89 ± 2.98,
p = 0.031) (figure
3). A significant relationship was also found between
PASI scores and both S. aureus cultivation and toxin production in
the lesional skin of the psoriasis patients (r = 0.315 and r =
0.316, respectively, p < 0.05). However, no significant
correlation was found between PASI scores and cultivation or toxin
production in non-lesional skin or nares in patients with psoriasis
(p > 0.05).
Table 2 The distribution of superantigens in the study
subjects
|
Superantigens
|
Healthy controls
|
Psoriasis patients
|
|
Nares
|
Normal skin
|
Nares
|
Nonlesional skin
|
Lesional skin
|
|
sea
|
3
|
0
|
15
|
2
|
19
|
|
seb
|
1
|
0
|
7
|
0
|
9
|
|
sec
|
0
|
0
|
5
|
0
|
7
|
|
sed
|
1
|
0
|
4
|
0
|
8
|
|
see
|
0
|
0
|
4
|
0
|
6
|
|
tst
|
0
|
0
|
3
|
1
|
4
|
|
eta
|
0
|
0
|
0
|
0
|
2
|
|
etb
|
0
|
0
|
11
|
1
|
18
|
|
mecA
|
3
|
0
|
0
|
0
|
7
|
|
Total
|
8
|
0
|
49
|
4
|
80
|
Discussion
In the present study, S. aureus was cultivated from lesional skin
and non-lesional skin in 64% and 14%, respectively, in patients
with psoriasis. This concurs with previous studies [8, 15]. The
cultivation in lesional skin was significantly higher than that in
non-lesional skin, whereas no significant difference was found
between the cultivation in normal skin of healthy controls and that
of the non-lesional skin of the psoriasis patients. Higher PASI
scores were detected in patients with cultivation-positive than in
patients with cultivation-negative skin. We also found a
significant relationship between PASI scores and both S. aureus
cultivation and toxin production in lesional skin of the patients
with psoriasis. These findings suggest that S. aureus colonisation
and toxin production are associated with psoriasis. Tomi et al.
isolated 36% toxigenic (sea, b, c and d) strains by using the latex
agglutination test in lesional skin of patients with psoriasis.
They reported significantly higher PASI scores in patients with
enterotoxin-positive psoriasis than in patients who were toxin
negative [8]. Sayama et al. investigated et, seb and tst-1 in
patients with psoriasis and found only 5 toxin producing S. aureus
in 100 patients in lesional skin. They suggested that superantigens
are not essential to maintain psoriasis [10].
We identified in 31 (96.8%) out of 32 patients at least one
superantigen positive strain in lesional skin, which represents a
rate higher than that reported in previous reports, but which may
explained by the fact that we investigated more toxins (9 toxins)
in all subjects. The strains cultivated from non-lesional skin, 3
(42.3%) out of 7 were toxigenic. There was a significant difference
between the toxigenic strains from lesional skin and those from
non-lesional skin (p = 0.01). The studies reported so far have
focused on the staphylococcal superantigens such as "classical"
enterotoxins a-d (sea-sed) and tst-1, which are found in varying
percentages on the skin of patients with psoriasis [8-10]. In the
present study, the toxin most often detected in lesional skin was
sea followed by etb, seb, sed, sec, mecA, see, tst and eta.
The present study is the first to investigate other toxins such
as mecA and see in patients with psoriasis. For the lesional skin,
mecA, etb, eta and see was identified in 7 (22.6%), 18 (58.1%), 2
(6.5%) and 6 (19.4%) patients, respectively, while at least one of
the “classical” superantigens like sea, seb, sec, sed or tst was
found in 28 (90.3%) strains. Three (9.7%) strains were positive for
mecA, etb or see but not for one of the classical superantigens.
These findings support a potential association of the mecA, etb and
see with psoriasis.
We also aimed to determine the frequencies of the nasal carriage
of S. aureus and its toxin production in patients with psoriasis
and to compare them with healthy controls. S. aureus is present as
a commensal in the nares of about 30% of healthy people [16]. In
the present study, S. aureus was cultivated from nares in 50% of
patients with psoriasis whereas from the nares of only 34% of
healthy controls (p > 0.05). Of the strains cultivated from
nares in patients with psoriasis, 96% (24/25) were toxigenic
whereas 41.2% (7/17) were toxigenic in healthy controls (p =
0.006). This suggests an association between toxigenic strains of
S. aureus and psoriasis. The nose and skin mostly produced same
strains in a given patient. Seventy-one percent (10/14) of the
toxigenic strains from both sites (lesional skin and nares)
produced the same toxins in a given patient. This supports the
hypothesis that S. aureus may spread over the skin surface by auto
transmission to the nose.
In conclusion, our results confirm the potential association
between S. aureus colonization and psoriasis. Toxigenic strains
isolated from both lesional skin and nares of the patients with
psoriasis were significantly higher than those of healthy controls.
A significant relationship between PASI scores and toxin
production was also demonstrated. Our results also showed that
non-classical superantigens such as mecA, etb and see may be
associated with psoriasis.
Acknowledgements
This study was supported by Mustafa Kemal University Research Fund.
Conflict of interest: None.
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